Treating iron deficiency with ferric carboxymaltose

ABSTRACT

The present invention relates to the field of treating iron deficiency with IV iron carbohydrate complexes such ferric carboxymaltose, monitoring or identifying subjects to determine their eligibility for being administered said IV iron carbohydrate complexes, and combining said IV iron carbohydrate complexes with additional drugs in order to mitigate or reduce side effects induced by said IV iron carbohydrate complexes.

This application is a divisional of Patent Cooperation Treaty (PCT)Appl. No. PCT/EP2019/079528 filed Oct. 29, 2019 which claims priority toEuropean Patent Application Nos. 18203223.5 filed Oct. 29, 2018 and18203818.2 filed Oct. 31, 2018. The disclosures of each and all of theaforementioned PCT and European applications are incorporated byreference in their entireties herein.

FIELD OF THE INVENTION

The present invention relates to the field of treating iron deficiencywith IV iron carbohydrate complexes, monitoring or identifying subjectsto determine their eligibility for being administered said IV ironcarbohydrate complexes, and combining said IV iron carbohydratecomplexes with additional drugs in order to mitigate or reduce sideeffects induced by the IV iron carbohydrate complexes.

BACKGROUND

Iron deficiency (ID) impairs the body's ability to produce hemoglobin,the key oxygen transporter, and impairs the function of key energy (ATP)producing enzymes. Symptoms consequently include fatigue and other signsof energy deprivation such as rapid heartbeat, shortness of breath, andchest pain.

ID has serious consequences. In chronic heart failure (CHF) patients,the risk of death or hospitalization is increased in patients with IDrelative to patients with normal iron status. Quality of life (QoL) isseverely affected and improves rapidly upon restoration of iron stores.Patients with ID or IDA undergoing surgery have poor outcomes—in partdue to greater risk of blood transfusions. Maternal iron deficiency isassociated with increased risk of pre-term birth and impaired fetalbrain development.

Iron deficiency anemia (IDA) develops when iron stores are depleted. Itis widespread. About 1 billion people worldwide suffer from IDAaccording to the WHO. 4.5 million patients are diagnosed with IDA Dailyoral iron is the first line therapy for most IDA patients but oftenfails due to lack of compliance, lack of efficacy and side effects.

High dose intravenous (IV) iron is an attractive treatment option.Patients typically require 1-3 grams of iron per year and high dose IViron effectively and rapidly improves symptoms and increases hemoglobinlevels. High dose IV iron allows treatment in one or few visits and IViron is the only option for patients failing oral iron.

Ferric carboxymaltose (FCM) belongs to a new generation of high dose IViron products. While older low dose products (ferric gluconate and ironsucrose) required 5-20 visits, these new generation products allow foriron correction in one or two visits by fast infusion of the product.

Moderate and transient decreases in serum phosphate (S-phosphate) havebeen observed for all iron complexes upon IV administration in humans totreat iron deficiency or iron deficiency anemia. This general phenomenonis believed by some to be associated with the consumption of phosphatein erythropoiesis, a primary intended effect of parenteral iron therapy.See, for instance, Van Wyck et al., 2009. Others have favored a theoryof phosphate wasting which postulates that the renal phosphate losscould be the consequence of proximal tubular dysfunction due to a directtoxic effect of IV iron on proximal renal tubular cells. Prats et al.,2013.

Ferric carboxymaltose (FCM) is a very commonly used iron carbohydratecomplex to treat patients with ID or IDA who are not on dialysis. It iscommercially available in the United States under the tradenameInjectafer® and in the European Union and many other countries under thetradename Ferinject®. A typical treatment regimen of FCM consists eitherof two doses of 750 mg of elemental iron given as intravenous infusionone week apart (this is the approved use according to the US label) oras an infusion of 1000 mg of elemental iron followed by an additionaldose of 500-1000 mg one week after (this is the approved use accordingto its EU label).

FCM has been shown to lead to a larger and longer reduction in serumphosphate as compared to iron dextran (Wolf et al., 2013), iron sucrose(WO2013/134273 A1) and iron isomaltoside 1000 (Bager et al., 2016,Schaefer et al., 2016, Zoller et al., 2017) and as a result a higherprevalence of hypophosphatemia, i.e., the condition characterized by toolow serum phosphate.

Nonetheless, although there are individual case reports onhypophosphatemia resulting from the treatment with FCM (Anand G, SchmidC, BMJ Case rep 2017) and subsequent bone complications such asosteomalacia occurring months thereafter (see, for instance, Schaefer etal. 2017; Klein et al. 2018), such findings have been actively disputedby the majority of publications or characterized as so rare to be acuriosity. A large number of scientific publications on the use of FCMdescribe how the associated hypophosphatemia is considered to betransient, asymptomatic and/or clinically irrelevant. See, for instance,Aksan et al., 2007; Bregman et al., 2014; Charytan et al., 2013;Evstatiev, 2011; Hussain et al., 2013; Ikuta etal., 2018; Prats et al.,2013; Qunibi et al., 2011; Sari et al., 2017; Seid et al., 2008; Steinet al., 2018; Van Wyck et al., 2009. This perspective has so far beensupported by the absence of studies demonstrating any short-termclinical impacts of the lowered phosphate levels despite the changes inbiochemical parameters.

US and EU regulators have so far also taken the position thatFCM-associated hypophosphatemia is transient, asymptomatic andclinically irrelevant. Although the U.S. FDA in a 2007 non-approvalletter listed clinically important hypophosphatemia as one of threepotential safety risks that would need to be resolved through additionalclinical data in order to verify the safety of the product, thesubsequent submission of data by the sponsor of Injectafer® led theagency to conclude in 2013 that all the clinical (efficacy and safety)issues brought forth in the non-approval letter including the issue ofclinically important hypophosphatemia had been satisfactorily resolved(U.S. Federal Drug Administration Center for drug evaluation andresearch, application number: 2035650rig1s000, summary review 2013). Theview that hypophosphatemia associated with FCM is mild and transient isalso reflected in the currently approved labels, which listhypophosphatemia as a side effect, but do not provide particularwarnings related to hypophosphatemia, neither in terms of short- orlong-term consequences. On the contrary, aside from listing it as a sideeffect the only mention of hypophosphatemia in the EU Summary of ProductCharacteristics (SmPC) which forms part of the regulatory approval ofFCM in Europe is the following statement: “In clinical trials, theminimum serum phosphorous values were obtained after approximately 2weeks, and 4 to 12 weeks following Ferinject treatment the values hadreturned to those within the range of baseline”.

Fibroblast growth factor 23 (FGF23) is an osteocyte-derived hormone thatregulates phosphate and vitamin D homeostasis. It undergoes proteolyticcleavage and as a result a mix of uncleaved, i.e. intact FGF23 (iFGF23),and its cleavage fragments are found in vivo. Because reduced serumphosphate in response to intravenous iron was suggested to be mediatedby an acute increase in FGF23, Wolf et al. examined the effects of irondeficiency and its rapid correction on C-terminal and intact FGF23levels in women with iron deficiency anemia secondary to heavy uterinebleeding. Their findings suggested that iron deficiency increasesC-terminal FGF23 (cFGF23) levels, and that FCM temporarily increasediFGF23 levels and reduced serum phosphate. Wolf et al., 2013;WO2013/134273 A1.

We have surprisingly found that contrary to the general understanding inthe art, treatment with FCM according to current practice leads todirect clinical consequences such as reduced muscle function andincreased bone turnover. Furthermore, we have found that the currentpractice related to repeated dosing of FCM one week apart leads to anauto-synergistic impact on iFGF23, with the second dose leading to a 2-3fold higher increase than the first dose.

Based on this understanding, there is clearly a need for improvedmethods of using FCM in the treatment of the underlying ID or IDA, whichmethods substantially decrease the risk of iFGF23-induced consequencessuch as reduced muscle function and increased bone turnover. These andother iFGF23-induced metabolic, nutritional and musculo-skeletalconsequences of FCM treatment are hereinafter referred to as theiFGF23-mediated or iFGF23-induced side effects.

SUMMARY

In one aspect of this invention, the treatment with ferriccarboxymaltose can be completed without loss of efficacy, but with areduced risk of iFGF23-mediated side effects by adjusting the timingand/or the amount of FCM administered in order to avoid auto-synergisticeffects.

In a second aspect of this invention, patients are selected fortreatment with ferric carboxymaltose not only based on the criteriacommonly used to define eligibility for IV iron, i.e. diagnosis of ID orIDA and a potential lack of the ability to tolerate or absorb oral iron,but also based on being less likely to suffer from iFGF23-mediated sideeffects.

In a third aspect of this invention, a subject who has been administereda first dose of ferric carboxymaltose is monitored to determine if orwhen the subject is eligible for being administered a second dose offerric carboxymaltose.

In a fourth aspect of this invention, a subject having a reduced riskfor FGF23-mediated side effects is identified.

In a fifth aspect of this invention, ferric carboxymaltose is combinedwith supporting drugs to mitigate or reduce the impact ofiFGF23-mediated side effects.

In line with these aspects, the present invention in particular relatesto therapeutic methods of treating iron deficiency which compriseadministering ferric carboxymaltose according to defined regimens and/orto selected subgroups of subjects; diagnostic methods for monitoringsubjects who have been administered a first dose of FCM to adjust thetiming and/or the amount of further FCM administration, or foridentifying subjects suitable for the therapeutic methods of theinvention; and combinations of FCM with other drugs that mitigate orreduce the impact of iFGF23-mediated side effects.

In a first embodiment of said first aspect, the present inventionrelates to a method of treating iron deficiency, which comprisesadministering a first dose and a second dose of ferric carboxymaltose,wherein the time between the first and the second dose is at least 10days.

In a second embodiment of said first aspect, the present inventionrelates to a method of treating iron deficiency, which comprisesadministering a first dose and a second dose of ferric carboxymaltose,wherein the first and the second dose each do not exceed 500 mg ofelemental iron.

In a third embodiment of said first aspect, the present inventionrelates to a method of treating iron deficiency, which comprisesadministering one or more doses of ferric carboxymaltose, wherein thetotal amount of elemental iron administered within a period of 12 monthsdoes not exceed 5000 mg.

In a first embodiment of said second aspect, the present inventionrelates to a method of treating iron deficiency which comprisesadministering ferric carboxymaltose, wherein the subject having areduced risk for FGF23-mediated side effects has blood parameters asdisclosed herein.

In a second embodiment of said second aspect, the present inventionrelates to a method of treating iron deficiency which comprisesadministering ferric carboxymaltose, wherein the subject having areduced risk for FGF23-mediated side effects is characterized by theabsence of exclusion criteria as disclosed herein.

In a third embodiment of said second aspect, the present inventionrelates to a method of treating iron deficiency which comprisesadministering ferric carboxymaltose, wherein the subject having areduced risk for FGF23-mediated side effects is characterized byrespiratory capacity as disclosed herein.

In a first embodiment of said third aspect, the present inventionrelates to a method of monitoring a subject who has been administered afirst dose of ferric carboxymaltose, comprising determining in abiological sample obtained from the subject at least one blood or urineparameter selected from the group consisting of (1) serum phosphatelevel, (2) serum vitamin D level, (3) serum ionized calcium level, (4)serum PTH level and (5) fractionary urinary phosphate excretion, whereinthe subject is eligible for being administered a second dose of ferriccarboxymaltose if the at least one blood or urine parameter is asdisclosed herein.

In a second embodiment of said third aspect, the present inventionrelates to a method of monitoring a subject who has been administered afirst dose of ferric carboxymaltose, comprising determining in abiological sample obtained from the subject at least one blood parameterselected from the group consisting of (1) serum Bone Specific AlkalinePhosphatase level; (2) serum Alkaline Phosphatase level, (3) serumN-terminal Propeptide of Type I Collagen (PINP) level and (4) serumCarboxy-terminal Collagen Crosslinks (CTx) level, wherein the subject iseligible for being administered a second dose of ferric carboxymaltoseif the at least one blood parameter is as disclosed herein.

In a third embodiment of said third aspect, the present inventionrelates to a method of monitoring a subject who has been administered afirst dose of ferric carboxymaltose, comprising determining therespiratory capacity of the subject, wherein the subject is eligible forbeing administered a second dose of ferric carboxymaltose if therespiratory capacity is as disclosed herein.

In a first embodiment of said fourth aspect, the present inventionrelates to a method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining in a biologicalsample obtained from the subject at least one blood or urine parameterselected from the group consisting of (1) serum phosphate level, (2)serum vitamin D level, (3) serum ionized calcium level, (4) serum PTHlevel and (5) fractionary urinary phosphate excretion, wherein thesubject has a reduced risk for FGF23-mediated side effects if the atleast one blood or urine parameter is as disclosed herein.

In a second embodiment of said fourth aspect, the present inventionrelates to a method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining in a biologicalsample obtained from the subject at least one blood parameter selectedfrom the group consisting (1) serum Bone Specific Alkaline Phosphataselevel; (2) serum Alkaline Phosphatase level, (3) serum N-terminalPropeptide of Type I Collagen (PINP) level and (4) serumCarboxy-terminal Collagen Crosslinks (CTx) level, wherein the subjecthas a reduced risk for FGF23-mediated side effects if the at least oneblood parameter is as disclosed herein.

In a third embodiment of said fourth aspect, the present inventionrelates to a method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining whether the subjectis characterized by one or more and in particular all of the followingexclusion criteria:

(1) having undergone bariatric surgery;

(2) obesity;

(3) cardiac conditions with increased risks of arrhythmias;

(4) primary or secondary hyperparathyroidism;

(5) pulmonary disorders such as asthma or chronic obstructive pulmonarydisease (COPD)

(6) genetic diseases leading to hypophosphatemia such as X-linkedhypophosphatemia, autosomal dominant hypophosphatemic rickets, autosomalrecessive hypophosphatemic rickets;

(7) secondary hypophosphatemia or tumor induced hypophosphatemia;

(8) disorders of the bone, such as for example osteoporosis orosteomalacia; and

(9) being scheduled for surgery within 1 day to two months of the ironadministration, wherein the subject has a reduced risk forFGF23-mediated side effects if the subject is characterized by theabsence of one or more and in particular all of the exclusion criteria.

In a fourth embodiment of said fourth aspect, the present inventionrelates to a method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining the respiratorycapacity of the subject, wherein the subject has a reduced risk forFGF23-mediated side effects if the respiratory capacity is as disclosedherein.

In a first embodiment of said fifth aspect, the present inventionrelates to a combination of ferric carboxymaltose with one or moreadditional drugs selected from the group consisting of:

(1) vitamin Ds, such as calcitriol;

(2) phosphates, such as glucose-1-phosphate, calcium phosphate or sodiumphosphate; and

(3) anti-FGF23 antagonistic antibodies, such as burosumab.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a table summarizing the analysis of the occurrence ofhypophosphatemia (primary endpoint) for study IDA-04.

FIG. 2 is a table summarizing the analysis of the occurrence ofhypophosphatemia (primary endpoint) for study IDA-05.

FIG. 3 shows the incidence of phosphate levels ≤2.0 mg/dL by visit(IDA-04 and IDA-05 combined; FCM: left-hand bars, right-hand bars).

FIG. 4 shows the incidence of phosphate levels ≤1.0 mg/dL by visit(IDA-04 and IDA-05 combined; FCM: left-hand bars, IIM: right-hand bars).

FIG. 5 shows the mean absolute phosphate levels over time (IDA-04 andIDA-05 combined).

FIG. 6 shows the change in iFGF23 over time (IDA-04).

FIG. 7 shows the change in iFGF23 over time (IDA-05).

FIG. 8 shows the change in cFGF23 over time (IDA-04).

FIG. 9 shows the change in cFGF23 over time (IDA-05).

FIG. 10 shows the change of fractional urinary phosphate excretion overtime (IDA-04 and IDA-05 combined).

FIG. 11 shows the mean change from baseline and absolute change ofvitamin D levels over time (IDA-04 and IDA-05 combined).

FIG. 12 shows the change from baseline and absolute change of intactparathyroid hormone levels over time (IDA-04 and IDA-05 combined).

FIG. 13 shows the change from baseline and absolute change of calciumions over time (IDA-04 and IDA-05 combined).

FIG. 14 shows the mean change of several makers of muscle function andbone turnover over time (IDA-04 and IDA-05 combined).

FIG. 15 shows the mean change in respiratory muscle strength over time(IDA-04 and IDA-05 combined).

DETAILED DESCRIPTION

Described herein are therapeutic methods of treating iron deficiencywhich comprise administering an iron carbohydrate complex, methods ofmonitoring a subject who has been administered a first dose of an ironcarbohydrate complex, methods of identifying a subject suitable for thetherapeutic methods of the invention, and combinations of an ironcarbohydrate complex with additional drugs, wherein the ironcarbohydrate complex induces a significant (e.g., statisticallysignificant) increase of iFGF23 levels in subjects under treatment. Themethods of the invention are thus applicable to complexes that share themechanism of inducing significant increases in iFGF23 and which, as aresult, can reduce serum phosphate levels and thus lead tohypophosphatemia. While this does not include the other IV iron drugscommonly used in Europe and the US, (such as iron sucrose (Venofer®),iron gluconate (Ferrlecit®), iron isomaltoside 1000 (Monofer®), or irondextran (Cosmofer®/INFeD® and Dexferrum®), it does include certaincomplexes available in Asia-Pacific including iron saccharated oxideavailable in Japan as Fesin® (which despite similarity in name isdistinct from iron sucrose in Venofer®), some species of ironpolymaltose and above all ferric carboxymaltose (FCM). Results fromclinical trials and case reports suggest that the highest risk for aniFGF23-mediated reduction of serum phosphate levels and the developmentof hypophosphatemia is associated with iron polymaltose, saccharatediron oxide, and above all ferric carboxymaltose. See, for instance, Wolfet al., 2013.

Accordingly, the preferred iron carbohydrate complex of this inventionis ferric carboxymaltose (FCM). The term “ferric carboxymaltose” as usedherein refers to colloidal complexes comprising iron, e.g., as ironoxide hydroxide, and carboxymaltose. Carboxymaltose is based on starchor starch derivatives that have been carboxylated, i.e., modified toinclude carboxy groups, for example through oxidation of the aldehydeend groups. A particular ferric carboxymaltose is obtainable byoxidizing maltodextrin, as described, for instance, in WO2007/081744 A1as VIT-45 and WO2004/037865 A1. A preferred example of ferriccarboxymaltose is commercially available in the United States under thetradename Injectafer® and in the European Union and many other countriesunder the tradename Ferinject®.

Definitions

In order that the present description may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

“Treatment” or “therapy” of a subject refers to any type of interventionor process performed on, or the administration of an active agent to,the subject with the objective of reversing, alleviating, ameliorating,inhibiting, slowing down, or preventing the onset, progression,development, severity, or recurrence of a symptom, complication,condition, or biochemical indicia associated with a disease.

A “subject” includes any human or nonhuman animal. The term “nonhumananimal” includes, but is not limited to, vertebrates such as nonhumanprimates, sheep, dogs, and rodents such as mice, rats and guinea pigs.In preferred embodiments, the subject is a human. The terms, “subject”and “patient” are used interchangeably herein.

A “therapeutically effective amount” or “therapeutically effective dose”of a drug or therapeutic agent is any amount of the drug that, when usedalone or in combination with another therapeutic agent, protects asubject against the onset of a disease or promotes disease regressionevidenced by a decrease in severity of disease symptoms, an increase infrequency and duration of disease symptom-free periods, or a preventionof impairment or disability due to the disease affliction. The abilityof a therapeutic agent to promote disease regression can be evaluatedusing a variety of methods known to the skilled practitioner, such as inhuman subjects during clinical trials, in animal model systemspredictive of efficacy in humans, or by assaying the activity of theagent in in vitro assays.

“Fibroblast growth factor 23 (FGF23)” is an osteocyte-derived hormonethat regulates phosphate and vitamin D homeostasis. FGF23 undergoesproteolytic cleavage and as a result a mix of uncleaved, i.e. intactFGF23 (iFGF23), and its cleavage fragments are found in vivo. The intactform, iFGF23, is the active form in relation to phosphate metabolismwhere it controls the urinary excretion of phosphate, with increasinglevels of iFGF23 leading to urinary wasting of phosphate. Two main typesof antibody assays currently exist, one which captures only iFGF23 andanother which binds to the C-terminal end of the hormone and thereforecaptures both iFGF23 and C-terminal fragments. The latter metric,cFGF23, is therefore a measure of the sum of intact FGF23 and C-terminalFGF23 fragments. Thus, two test related to FGF23 exist, iFGF23 andcFGF23, which have different interpretations.

“Hypophosphatemia” is a condition characterized by too low serumphosphate levels. The Common Terminology Criteria for Adverse Events(CTCAE) Version 4.0 provides four grades of hypophosphatemia.

Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 <LLN-2.5 <2.5-2.0 <2.0-1.0 <1.0Death mg/dL mg/dL mg/dL mg/dL <LLN-0.8 <0.8-0.6 <0.6-0.3 <0.3 mmol/Lmmol/L mmol/L mmol/L LLN is the lower limit of normal range used by aspecific laboratory.

As used herein, unless specified otherwise, frequencies ofhypophosphatemia refer to serum phosphate levels below 2 mg/dL.

The term “serum phosphate (S-phosphate)” as used herein refers to thelevel of inorganic phosphorus in serum blood as measured as an ammoniumphosphomolybdate complex having the formula (NH₄)₃[PO₄](MoO₃)₁₂ formedby the reaction of inorganic phosphorous with ammonium molybdate in thepresence of sulfuric acid. The complex is determined photometrically inthe ultraviolet region (340 nm) of the spectrum using the Roche Modularand Cobas Analyzers.

The term “serum vitamin D” as used herein refers to the level of vitaminDs, in particular 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, and24,25-dihydroxyvitamin D, in blood serum as measured by LiquidChromatography and Tandem Mass Spectrometry (LC-MS/MS).

The term “serum ionized calcium” as used herein refers to the level ofcalcium ions (Ca²) in blood serum as measured using the IL GEM Premier3500 PAK cartridge. The central component is the sensor card, whichprovides a low volume, gas tight chamber in which the blood sample ispresented to the sensors. The pH and electrolyte sensors are all basedon the principle of ion-selective electrodes; that is, an electricalpotential can be established across a membrane which is selectivelypermeable to a specific ion. The pH and electrolyte sensors arepolyvinyl chloride (PVC) based ion selective electrodes, consisting ofan internal Ag/AgCl reference electrode and internal salt layer. Thepotentials are measured against the card reference electrode.

The term “serum parathyroid hormone (PTH)” as used herein refers to thelevel of serum parathyroid hormone in blood serum as measured using atwo-site sandwich immunoassay using direct chemiluminometric technology,which used constant amounts of two anti-human PTH antibodies. PTH,produced by the parathyroid gland, is the major circulating factorregulating extracellular calcium concentration. Abnormally low ionizedcalcium concentrations trigger the secretion of PTH. The PTH moleculesbind to type 1 PTH receptors in target tissues and initiate a sequenceof reactions that results in an increase in extracellular calciumconcentrations. PTH stimulates osteoclastic bone resorption resulting inthe release of calcium from bone. PTH stimulates transcellular calciumreabsorption from the renal tubules and stimulates the kidney to produce1,25-dihydroxyvitamin D, which acts on the intestines to increasecalcium reabsorption. In most clinical conditions, rising levels ofextracellular calcium will suppress PTH secretion through a negativefeedback mechanism.

The term “fractionary urinary excretion of phosphate (FEPi)” (sometimesabbreviated FEPO4) is a measure of how much phosphate is not re-absorbedfrom the pre-urine in the kidneys, i.e., how much phosphate ends up inthe urine as a proportion of how much phosphate the subject has in theblood. It is calculated like this: FEPi=[PO4 (Urine)*Creatinine(Serum)]/[PO4 (Serum)*Creatinine (Urine)]*100. An FEPi of 10-20%(0.1-0.2 fraction) is usually considered to be normal; an FEPi<10% (<0.1fraction) is usually considered to be low; and an FEPi>20% (>0.2fraction) is usually considered to be high. Phosphate in urine ismeasured using the same methodology as for serum.

The term “serum Bone Specific Alkaline Phosphatase” as used hereinrefers to the level of Bone specific Alkaline Phosphatase in blood serumas measured using the Beckman-Coulter Ostase assay which is a one-stepimmunoenzymatic chemiluminescence assay using a mouse monoclonalantibody specific to Bone Specific Alkaline Phosphatase (BAP).

The term “serum Alkaline Phosphatase” as used herein refers to the levelof Alkaline Phosphatase in blood serum as measured enzymatically using aRoche Modular Analyzer. In the presence of magnesium and zinc ions,p-nitrophenyl phosphate is cleaved by phosphatase into phosphate andp-nitrophenol. The p-nitrophenol released is proportional to theAlkaline Phosphatase activity and is measured photometrically.

The term “serum N-terminal Propeptide of Type I Collagen (PINP)” as usedherein refers to the level of PINP in blood serum as measured using asandwich principle, electrochemiluminescence immunoassay (ECLIA) on aCobas e601 Analyzer. During the first incubation, PINP in the sample anda biotinylated monoclonal PINP-specific antibody are incubated together.During the second incubation, streptavidin-coated labelledmicroparticles and a monoclonal PINP-specific antibody labelled withruthenium complex (Trs (2,2-bipyridyl)ruthenium (II)-complex(Ru(bpy)23+)) are added to form a sandwich complex, which binds to thesolid phase via interaction of biotin and streptavidin. The reactionmixture is aspirated into the measuring cell where the microparticlesare magnetically captured onto the surface of the electrode. A voltageis applied to the electrode, which induces chemiluminescent emission,which is measured by a photomultiplier. Results are determined via acalibration curve, which is instrument-specifically generated by a2-point calibration and a master curve provided via the reagent barcode.

The term “serum Carboxy-terminal Collagen Crosslinks (CTx)” as usedherein refers to the level of CTx in blood serum as measured using asandwich principle, electrochemiluminescence immunoassay (ECLIA) on aCobas e601 Analyzer. During the first incubation, 50 μL of sample andbiotinylated monoclonal anti-beta-CrossLaps antibodies are incubatedtogether. During the second incubation, streptavidin-coated labelledmicroparticles and a monoclonal P-beta-CrossLaps-specific antibodylabelled with ruthenium complex are added to form a sandwich complex,which binds to the solid phase via interaction of biotin andstreptavidin. The reaction mixture is aspirated into the measuring cellwhere the microparticles are magnetically captured onto the surface ofthe electrode. Unbound substances are then removed with ProCell. Avoltage is applied to the electrode, which induces chemiluminescentemission, which is measured by a photomultiplier. Results are determinedvia a calibration curve, which is instrument-specifically generated by a2-point calibration and a master curve provided via the reagent barcode.

The term “serum creatine kinase (CK)” as used herein refers to the CKlevel in blood serum as measured enzymatically using a Roche Modular andCobas Analyzers. CK catalyzes the phosphorylation of ADP by creatinephosphate. ATP is performed which phosphorylates glucose and theresulting glucose-6-phosphate converts NADP+ to NADPH. The rate of NADPHformation is proportional to CK activity and is measuredphotometrically. CK is an enzyme that catalyzes the reversible transferof phosphate from ATP to Creatine. This makes possible the storage ofhigh-energy phosphate in a more stable form in ATP. CK is present inhigh concentration in skeletal muscle, cardiac muscle, thyroid, prostateand brain; it is present only in small amounts in liver, kidney, lungand other tissues. Hence, an increase in serum CK activity is ascribedprimarily to damage to striated muscle (skeletal or cardiac) and in rarecases, to brain. Differentiation of these various diseases canfrequently be made upon clinical grounds, but there are situations whenthis is not possible. Measurement of CK isoenzymes helps solve theproblem.

The term “serum Ferritin” as used herein refers to the level of Ferritinin blood serum as measured using a two-site immunoenzymatic (“sandwich”assay). Ferritin is the major iron storage protein for the body. Theconcentration of ferritin is directly proportional to the total ironstores of the body, resulting in serum ferritin levels becoming a commondiagnostic tool in the evaluation of iron status. Patients with irondeficiency anemia have serum ferritin levels approximately one tenth ofnormal subjects, while patients with iron overload (hemochromatosis,hemosiderosis) have serum ferritin level much higher than normal.Ferritin levels also provide a sensitive means of detecting irondeficiency at an early stage. In both adults and children, chronicinflammation results in a disproportionate increase in ferritin levelsin relation to iron reserves. Elevated ferritin levels also are observedin acute and chronic liver disease, chronic renal failure and in sometypes of neoplastic disease.

It is noted that while the above blood parameters are determined inserum, they can likewise be determined in plasma. Serum and plasmalevels correlate and can be converted into each other.

The term “bariatric surgery” as used herein refers to a surgicalprocedure aiming at introducing weight loss by reducing the size of thestomach with a gastric band or removal of a portion of the stomach(sleeve gastrectomy or biliopancreatic diversion with duodenal switch)or by resecting and re-routing the small intestine to a small stomachpouch.

“Obesity” is a condition characterized by excessive body weight to theextent when the body mass index (BMI), a measurement obtained bydividing a person's weight by the square of the person's height, is over30 kg/m².

“Cardiac conditions with increased risks of arrhythmias” are conditionscharacterized by cardiovascular conditions and risk factors thatincrease the chance of developing arrhythmias, or abnormal heart rhythmsincluding but not limited to: Coronary artery disease, endocarditis,valvular heart disease, high blood pressure, diabetes and obesity.

“Primary or secondary hyperparathyroidism” is a condition characterizedby excessive secretion of parathyroid hormone.

“Asthma” is a condition characterized by a chronic lung disease thatinflames and narrows the airways and causes recurring periods ofwheezing, chest tightness, shortness of breath, and coughing.

“Chronic obstructive pulmonary disease (COPD)” is a conditioncharacterized by a chronic inflammatory lung disease that causesobstructed airflow from the lungs.

“X-linked hypophosphatemia” is a condition characterized by a hereditaryrenal phosphate-wasting disorder characterized by low levels ofphosphate in blood, rickets and/or osteomalacia, and diminished growth.

“Autosomal dominant or recessive hypophosphatemic rickets” is acondition characterized by bones that become soft and bend easily, dueto low levels of phosphate in the blood.

“Secondary hypophosphatemia” is a condition characterized by low levelsof phosphate in blood due to decreased oral intake or decreasedintestinal absorption.

“Tumor induced hypophosphatemia” is a condition characterized by lowlevels of phosphate in blood secondary to tumor, such as FGF23 producingtumors.

“Osteoporosis” is a condition characterized by a bone disease thatoccurs when the body loses too much bone, makes too little bone, orboth. As a result, bones become weak.

“Osteomalacia” is a condition characterized by the softening of thebones caused by impaired bone metabolism primarily due to inadequatelevels of available phosphate, calcium, and vitamin D, or because ofresorption of calcium. The impairment of bone metabolism causesinadequate bone mineralization.

A subject “being scheduled for surgery within 1 day to two months of theiron administration” is a subject who will have surgery within 1 day totwo months of the iron administration.

For the purpose of this text, when specifying a dose in mg or g of aniron carbohydrate complex, consistent with the practice in theliterature, the value refers to the amount of elemental iron provided inmg.

A. Therapeutic Methods

Described herein are therapeutic methods of treating iron deficiencywhich comprise administering ferric carboxymaltose according to definedadministration regimens and/or to selected subgroups of subjects.Accordingly, the present invention also relates to ferric carboxymaltosefor use in said methods, the use of ferric carboxymaltose for treatingiron deficiency and or the use of ferric carboxymaltose in themanufacture of a medicament for treating iron deficiency.

I. Administration Regimens

According to a first embodiment of this aspect of the invention, themethod of treating iron deficiency comprises repeated administration ofthe iron carbohydrate complex, in particular FCM, which is characterizedin that the time between the first and the second dose is at least 10days. For instance, if the first dose is administered at day 0, thesecond dose is administered at day 10 or thereafter. Preferably, thetime between the first and the second dose is at least 14 days, 18 days,21 days, 28 days, 35 days, 42 days, 49 days, or 56 days. In some cases,it can even be expedient that the time between the first and the seconddose is at least 3 months or 6 months.

The daily dose of elemental iron administered is a therapeuticallyeffective amount that may be in the range of 500 mg to 1000 mg, e.g. at500 mg, 750 mg, or 1000 mg elemental iron.

The dose is in particular a single daily dose. For instance, a typicalsingle daily dose of ferric carboxymaltose is 500 mg, 750 mg or 1000 mgelemental iron. For repeated administration, a first dose of 750 mgelemental iron is followed by a second dose of 750 mg elemental iron, ora first dose of 1000 mg elemental iron is followed by a second dose of500 mg to 1000 mg of elemental iron, e.g. 500 mg, 750 mg or 1000 mg ofelemental iron. Further doses of FCM may follow.

According to a second embodiment of this aspect of the invention, themethod of treating iron deficiency comprises repeated administration ofthe iron carbohydrate complex, in particular FCM, which is characterizedin that each of the first and the second dose does not exceed, or isless than, 500 mg of elemental iron. Exemplary amounts include 500 mg,400 mg, 300 mg, 200 mg and 100 mg.

According to a third embodiment of this aspect of the invention, themethod of treating iron deficiency comprises the administration of theiron carbohydrate complex, in particular FCM, which is characterized inthat the total amount of elemental iron administered within a period of12 months does not exceed 5000 mg. Preferably, the total amount ofelemental iron administered within a period of 12 months does not exceed4000 mg, 3000 mg or 2000 mg.

II. Selected Subgroups of Subjects

The methods of the invention are typically performed on a subject inneed thereof. A subject in need of the methods of the invention is asubject having, diagnosed with, suspected of having, or at risk fordeveloping iron deficiency, in particular, iron deficiency associatedwith chronic blood loss, acute blood loss, pregnancy, childbirth,lactation, childhood development, heavy uterine bleeding, menstruation,gastrointestinal bleeding, chronic internal bleeding, inflammatory boweldisease, congestive heart failure, restless leg syndrome, parasiticinfections, lost or impaired kidney function such as due to chronickidney disease or kidney failure, dialysis, surgery, chronic ingestionof agents such as alcohol, salicylates, steroids, non-steroidalanti-inflammatory agents, erythropoiesis stimulating agents (ESAs) ordrugs inhibiting iron absorption. Iron deficiency anemia (IDA) developswhen iron stores are depleted. Patients who suffer from ID may have IDA;patients with IDA necessarily suffer from ID. Methods to diagnose ID anIDA are well established in the art and commonly used in clinicalpractice.

Subjects having, diagnosed with, suspected of having, or at risk fordeveloping iron deficiency will be given IV iron in the form of an ironcarbohydrate complex, in particular ferric carboxymaltose, if oral ironis not tolerated or not effective in the subject. Another situationwhere IV iron is indicated is a need to deliver iron rapidly.

A particular group of subjects that are amenable to treatment accordingto the present invention is characterized as having a reduced risk forFGF23-mediated side effects.

According to a first embodiment of this aspect of the invention, asubject having a reduced risk for FGF23-mediated side effects has ablood or urine parameter selected from the group consisting of:

(1) normal serum phosphate level, in particular >2.5 mg/dL;

(2) normal serum vitamin D level, in particular 1,25-dihydroxyvitamin Dbeing within the following ranges: Males: <16 years: 24-86 pg/mL, ≥16years: 18-64 pg/mL, Females: <16 years: 24-86 pg/mL, ≥16 years: 18-78pg/mL;

(3) normal serum ionized calcium level, in particular 1.16-1.32 mmol/L;

(4) normal serum PTH level, in particular 15-65 pg/mL;

(5) normal fractionary urinary phosphate excretion, in particular anFEPi of 10-20% (0.1-0.2 fraction); and

(6) a combination of (1), (2), (3), (4), and (5).

According to a second embodiment of this aspect of the invention, asubject having a reduced risk for FGF23-mediated side effects has ablood parameter selected from the group consisting of:

(1) normal serum Bone Specific Alkaline Phosphatase level, in particular6.5-22.4 U/L;

(2) normal serum Alkaline Phosphatase level, in particular 31-140 U/L;

(3) normal serum N-terminal Propeptide of Type I Collagen (PIMP) level,in particular 15.13-85.50 ng/mL;

(4) normal serum Carboxy-terminal Collagen Crosslinks (CTx) level, inparticular 0.03-1.01 ng/mL; and

(5) a combination of (1), (2), (3) and (4).

According to a third embodiment of this aspect of the invention, asubject having a reduced risk for FGF23-mediated side effects ischaracterized by the absence of one or more and in particular all of thefollowing exclusion criteria:

(1) having undergone bariatric surgery;

(2) obesity;

(3) cardiac conditions with increased risks of arrhythmias;

(4) primary or secondary hyperparathyroidism;

(5) pulmonary disorders such as asthma or chronic obstructive pulmonarydisease (COPD)

(6) genetic diseases leading to hypophosphatemia such as X-linkedhypophosphatemia, autosomal dominant hypophosphatemic rickets, autosomalrecessive hypophosphatemic rickets;

(7) secondary hypophosphatemia or tumor induced hypophosphatemia;

(8) disorders of the bone, such as for example osteoporosis orosteomalacia; and (9) being scheduled for surgery within 1 day to twomonths of the iron administration such as preferably being scheduled forsurgery within 1 to 3 weeks of iron administration.

According to a fourth embodiment of this aspect of the invention, asubject having a reduced risk for FGF23-mediated side effects ischaracterized by normal respiratory capacity measured as maximalrespiratory pressure and maximal inspiratory pressure, in particular amaximal respiratory pressure of: Males: ≥117−(0.83×age) cm H₂O, Females:≥95−(0.57×age) cm H₂O; and/or a maximal inspiratory pressure of: Males:≥62−(0.15×age) cm H₂O, Females: ≥62−(0.50×age) cm H₂O.

According to a fifth aspect, a subject having a reduced risk forFGF23-mediated side effects has both blood/urine parameters as disclosedherein and is characterized by the absence of exclusion criteria asdisclosed herein.

According to a sixth aspect, a subject having a reduced risk forFGF23-mediated side effects has blood/urine parameters as disclosedherein, is characterized by the absence of exclusion criteria asdisclosed herein, and is characterized by normal respiratory capacity asdisclosed herein.

A further particular group of subjects that are amenable to treatmentaccording to the present invention are subjects with chronic kidneydisease (CKD). Frequencies of hypophosphatemia following iron treatmentare much lower in patients with chronic kidney disease (CKD), who haveimpaired renal function and thus impaired ability to excrete phosphatein the urine and as a result tend to rather suffer fromhyperphosphatemia (too high phosphate). On the other hand, due to thetendency towards hyperphosphatemia, CKD patients tend to have very highFGF23 levels function due to ongoing attempts of the body to compensatefor the high serum phosphate levels by producing iFGF23 to increase theurinary fractional excretion of phosphate via the kidneys. For thisreason, iFGF23 levels are elevated in CKD patients, and as a resultother downstream effects of iFGF23 may be more pronounced despite thelower risk of hypophosphatemia.

B. Diagnostic Methods

Further described herein are methods of monitoring a subject who hasbeen administered a first dose of an iron carbohydrate complex,comprising determining in a biological sample obtained from the subjectat least one blood or urine parameter selected from the group consistingof (1) serum phosphate level, (2) serum vitamin D level, (3) serumionized calcium level, (4) serum PTH level and (5) fractionary urinaryphosphate excretion, wherein the subject is eligible for beingadministered a second dose of the iron carbohydrate complex if the atleast one blood or urine parameter is as disclosed herein.

Further described herein are methods of monitoring a subject who hasbeen administered a first dose of ferric carboxymaltose, comprisingdetermining in a biological sample obtained from the subject at leastone blood parameter selected from the group consisting of (1) serum BoneSpecific Alkaline Phosphatase level; (2) serum Alkaline Phosphataselevel, (3) serum N-terminal Propeptide of Type I Collagen (PINP) leveland (4) serum Carboxy-terminal Collagen Crosslinks (CTx) level, whereinthe subject is eligible for being administered a second dose of ferriccarboxymaltose if the at least one blood parameter is normal asdisclosed herein.

Still further described herein are methods of monitoring a subject whohas been administered a first dose of ferric carboxymaltose, comprisingdetermining the respiratory capacity of the subject, wherein the subjectis eligible for being administered a second dose of ferriccarboxymaltose if the respiratory capacity is normal as disclosedherein.

Also described herein are methods of identifying a subject suitable forthe therapeutic methods of the invention. Such methods include methodsof identifying a subject having a reduced risk for FGF23-mediated sideeffects, comprising determining in a biological sample obtained from thesubject at least one blood or urine parameter selected from the groupconsisting of (1) serum phosphate level, (2) serum vitamin D level, (3)serum ionized calcium level, (4) serum PTH level and (5) fractionaryurinary phosphate excretion, wherein the subject has a reduced risk forFGF23-mediated side effects if the at least one blood or urine parameteris normal as disclosed herein.

Further described herein are methods of identifying a subject having areduced risk for FGF23-mediated side effects, comprising determining ina biological sample obtained from the subject at least one bloodparameter selected from the group consisting (1) serum Bone SpecificAlkaline Phosphatase level; (2) serum Alkaline Phosphatase level, (3)serum N-terminal Propeptide of Type I Collagen (PINP) level and (4)serum Carboxy-terminal Collagen Crosslinks (CTx) level, wherein thesubject has a reduced risk for FGF23-mediated side effects if the atleast one blood or urine parameter is normal as disclosed herein.

Still further described herein are methods of identifying a subjecthaving a reduced risk for FGF23-mediated side effects, comprisingdetermining whether the subject is characterized by one or more and inparticular all of the following exclusion criteria:

(1) having undergone bariatric surgery;

(2) obesity;

(3) cardiac conditions with increased risks of arrhythmias;

(4) primary or secondary hyperparathyroidism;

(5) pulmonary disorders such as asthma or chronic obstructive pulmonarydisease (COPD)

(6) genetic diseases leading to hypophosphatemia such as X-linkedhypophosphatemia, autosomal dominant hypophosphatemic rickets, autosomalrecessive hypophosphatemic rickets;

(7) secondary hypophosphatemia or tumor induced hypophosphatemia;

(8) disorders of the bone, such as for example osteoporosis orosteomalacia; and

(9) being scheduled for surgery within 1 day to two months of the ironadministration, wherein the subject has a reduced risk forFGF23-mediated side effects if the subject is characterized by theabsence of one or more and in particular all of the exclusion criteria.

And yet described herein are methods of identifying a subject having areduced risk for FGF23-mediated side effects, comprising determining therespiratory capacity of the subject, wherein the subject has a reducedrisk for FGF23-mediated side effects if the respiratory capacity isnormal as disclosed herein.

The biological sample can be any sample obtained from a subject, whereinthe sample allows determining said blood or urine parameters. In someembodiments, the biological sample comprises a blood sample. In someembodiments, the biological sample comprises a plasma sample. Inpreferred embodiments, the biological sample comprises a serum sample.In other preferred embodiments, the biological sample comprises a urinesample. As the diagnostic methods are performed on a sample of thesubject, the methods are carried out ex vivo, in particular in vitro.

The biological sample can be collected and/or obtained by any methodknown in the art. In some embodiments, the biological sample is obtaineddirectly from a subject, e.g., by withdrawing the sample directly fromthe circulatory system of a subject. In other embodiment, the biologicalsample is obtained from a lab, wherein the lab, or a predecessor,previously obtained the biological sample directly from a subject. Insome embodiments, the biological sample is fresh, e.g., the sample hasnot been frozen or stored for an extended period of time. In otherembodiments, the biological sample has been stored at a temperature lessthan 37° C.

The diagnostic methods of monitoring and identifying a subject accordingto the invention, in some embodiments, further comprises administeringthe iron carbohydrate complex, in particular ferric carboxymaltose, to asubject identified as being eligible of being administered a second doseof the iron carbohydrate complex and/or as having a reduced risk forFGF23-mediated side effects. The disclosure herein in relation to theadministration regimens and selected subgroups of patients is applicablein this regard.

C. Drug Combinations

Further described herein are combinations of FCM with one or moreadditional drugs for use in treating iron deficiency, wherein theadditional drug is selected from the group consisting of:

(1) vitamin Ds;

(2) phosphates; and

(3) anti-FGF23 antagonistic antibodies.

FCM leads to a decrease in active vitamin D, 1,25-dihydroxyvitamin D,and to an increase of 24,25-dihydroxyvitamin D. Administeringtherapeutically effective amounts of vitamin D can help reducing thiseffect. To this end, administration of alfacalcidol and in particularcalcitriol is preferred. Alternatively, cholecalciferol orergocalciferol may be administered.

If alfacalcidol or calcitriol is administered it may be administeredwithin three days prior to the administration of the first FCM dose.Alternatively, alfacalcidol or calcitriol administration is started onthe same day of first FCM dose. Still a further alternative is to startalfacalcidol or calcitriol administration on day 1, day 2, day 3, day 4,day 5, day 6 or day 7, or day 14 after the administration of the firstFCM dose.

Calcitriol is expediently administered in a daily dose of 0.125 μg to 2μg, such as 0.125 μg to 1 μg, such as 0.25 μg-0.75 μg, for example 0.50mg.

Alfacalcidol is expediently administered in daily doses of 0.25 μg to 5μg, such as 0.5 μg to 2 μg, such as 1 mg.

Whether administered as pre-treatment to FCM administration orsubsequent to FCM administration, treatment with calcitriol oralfacalcidol is continued for until blood parameters, in particularvitamin D level, are normal or until three weeks, four weeks, fiveweeks, or six weeks after initiation of treatment, whichever occursearlier.

Cholecalciferol or ergocalciferol is preferably administered prior totreatment with FCM, such as for a period of 14 days, 7 days, 6 days,alternatively 5 days, 4 days, 3 days, 2 days, 1 day prior toadministration of the first FCM dose.

Cholecalciferol is expediently administered in weekly doses from 140 μgto 2500 μg, such as preferably 300 μg-600 μg such as preferably 500 pg.

Ergocalciferol is expediently administered in daily doses of 10 μg to1250 μg, such as preferably 500 μg.

In one embodiment of the invention, the pre-treatment with eithercholecalciferol or ergocalciferol prior to administration of the firstFCM dose as disclosed herein is followed by treatment with alfacalcidolor calcitriol subsequent to administration of the first FCM dose asdisclosed herein.

Because FCM also leads to a decrease in serum phosphate, administeringtherapeutically effective amounts of phosphates can help reducing thiseffect. For example, glucose-1-phosphate or a phosphate salt such ascalcium phosphate, potassium phosphate or sodium phosphate can beadministered orally or intravenously (IV).

Therapeutically effective amounts of phosphate for IV administrationinclude single doses, preferably singly daily doses, of 10 mmol to 50mmol, such as 30 mmol to 40 mmol and, in particular, 15 mmol to 35 mmolof phosphate, which may be repeated until serum phosphate havenormalized.

Therapeutically effective amounts of phosphate for oral administrationinclude single doses, preferably singly daily doses, of 15 mmol to 85mmol, such as 30 mmol to 65 mmol and, in particular, 45 mmol to 50 mmolof phosphate, which may be repeated until serum phosphate havenormalized.

According to a particular embodiment of the invention, a combination ofadditional drugs is used. The combination of a vitamin D and a phosphateis a particularly preferred combination.

Anti-FGF23 antibodies are used to treat very rare diseases such asx-linked hypophosphatemia. The first such product to reach the market isburosumab. It is preferred that anti-FGF23 antibodies are used inpatients experiencing very severe FGF23 mediated side effects followingthe administration of FCM such as serum phosphate levels below 1 mg/dLor iFGF23 levels which are increased by 100 pg/mL or more relative tobaseline.

According to a preferred embodiment, the additional drug is administeredorally. This is in particular expedient if the additional drug is aphosphate, such as glucose-1-phosphate calcium phosphate or sodiumphosphate, or a vitamin D, such as calcitriol.

According to a particular embodiment, the additional drug isadministered prior to the first dose of FCM administration. This is inparticular expedient if the additional drug is cholecalciferol orergocalciferol.

According to another particular embodiment, the additional drug isadministered after the first dose but prior to the second dose of FCMadministration. This is in particular expedient if the additional drugis calcitriol or alfacalcidol. If phosphates used as additional drugs,it is likewise preferred to administer them after the first dose of FCMadministration.

Dosages for the additional drugs usually refer to amounts of drugadministered to adults. Dosages for administration to infants may beadjusted accordingly.

Exemplary Embodiments

1. A method of treating iron deficiency, which comprises administering afirst dose and a second dose of ferric carboxymaltose, wherein the timebetween the first and the second dose is at least 10 days.2. The method of embodiment 1, wherein the time between the first andthe second dose is at least 14 days, 18 days, 21 days, 28 days, 35 days,42 days, 49 days, or 56 days.3. The method of embodiment 1, wherein the time between the first andthe second dose is at least 3 months or 6 months.4. The method of any one of embodiments 1-3, wherein the first and thesecond dose is a single daily dose.5. The method of any one of embodiments 1-4, wherein the first dose is750 mg of elemental iron and the second dose is 750 mg of elementaliron.6. A method of treating iron deficiency, which comprises administering afirst dose and a second dose of ferric carboxymaltose, wherein the firstand the second dose each do not exceed, or are less than, 500 mg ofelemental iron.7. A method of treating iron deficiency, which comprises administeringone or more doses of ferric carboxymaltose, wherein the total amount ofelemental iron administered within a period of 12 months does not exceed5000 mg.8. A method of treating iron deficiency, which comprises administeringferric carboxymaltose to a subject, wherein the subject has a reducedrisk for FGF23-mediated side effects.9. The method of embodiment 8, wherein the subject having a reduced riskfor FGF23-mediated side effects has a blood or urine parameter selectedfrom the group consisting of:

(1) normal serum phosphate level, in particular >2.5 mg/dL;

(2) normal serum vitamin D level, in particular 1,25-dihydroxy vitamin Dbeing within the following ranges: Males: <16 years: 24-86 pg/mL, ≥16years: 18-64 pg/mL, Females: <16 years: 24-86 pg/mL, ≥16 years: 18-78pg/mL;

(3) normal serum ionized calcium level, in particular 1.16-1.32 mg/dL;

(4) normal serum PTH level, in particular 15-65 pg/mL;

(5) normal fractionary urinary phosphate excretion, in particular anFEPi of 10-20% (0.1-0.2 fraction); and

(6) a combination of (1), (2), (3), (4) and (5).

10. The method of embodiment 8, wherein the subject having a reducedrisk for FGF23-mediated side effects has a blood parameter selected fromthe group consisting of:

(1) normal serum Bone Specific Alkaline Phosphatase level, in particular6.5-22.4 U/L;

(2) normal serum Alkaline Phosphatase level, in particular 31-40 U/L;

(3) normal serum N-terminal Propeptide of Type I Collagen (PINP) level,in particular 15.13-85.50 ng/mL;

(4) normal serum Carboxy-terminal Collagen Crosslinks (CTx) level, inparticular 0.03-1.01 ng/mL; and

(5) a combination of (1), (2), (3) and (4).

11. The method of embodiment 8, wherein the subject having a reducedrisk for FGF23-mediated side effects is characterized by the absence ofone or more and in particular all of the following exclusion criteria:

(1) having undergone bariatric surgery;

(2) obesity;

(3) cardiac conditions with increased risks of arrhythmias;

(4) primary or secondary hyperparathyroidism;

(5) pulmonary disorders such as asthma or chronic obstructive pulmonarydisease (COPD)

(6) genetic diseases leading to hypophosphatemia such as X-linkedhypophosphatemia, autosomal dominant hypophosphatemic rickets, autosomalrecessive hypophosphatemic rickets;

(7) secondary hypophosphatemia or tumor induced hypophosphatemia;

(8) disorders of the bone, such as for example osteoporosis orosteomalacia; and

(9) being scheduled for surgery within 1 day to two months of the ironadministration.

12. The method of embodiment 8, wherein the subject having a reducedrisk for FGF23-mediated side effects is characterized by normalrespiratory capacity measured as maximal respiratory pressure and/ormaximal inspiratory pressure, in particular a maximal respiratorypressure of: Males: ≥117−(0.83×age) cm H₂O, Females: ≥95−(0.57×age) cmH₂O; and/or a maximal inspiratory pressure of: Males: ≥62−(0.15×age) cmH₂O, Females: ≥62−(0.50×age) cm H₂O.13. The method of any one of embodiments 1-7 for treating a subjecthaving a reduced risk for FGF23-mediated side effects as defined inembodiments 8-12.14. A method of monitoring a subject who has been administered a firstdose of ferric carboxymaltose, comprising determining in a biologicalsample obtained from the subject at least one blood or urine parameterselected from the group consisting of (1) serum phosphate level, (2)serum vitamin D level, (3) serum ionized calcium level, (4) serum PTHlevel and (5) fractionary urinary phosphate excretion, wherein thesubject is eligible for being administered a second dose of ferriccarboxymaltose if the at least one blood or urine parameter is asdefined in embodiment 9.15. A method of monitoring a subject who has been administered a firstdose of ferric carboxymaltose, comprising determining in a biologicalsample obtained from the subject at least one blood parameter selectedfrom the group consisting of (1) serum Bone Specific AlkalinePhosphatase level; (2) serum Alkaline Phosphatase level, (3) serumN-terminal Propeptide of Type I Collagen (PINP) level and (4) serumCarboxy-terminal Collagen Crosslinks (CTx) level, wherein the subject iseligible for being administered a second dose of ferric carboxymaltoseif the at least one blood parameter is as defined in embodiment 10.16. A method of monitoring a subject who has been administered a firstdose of ferric carboxymaltose, comprising determining the respiratorycapacity of the subject, wherein the subject is eligible for beingadministered a second dose of ferric carboxymaltose if the respiratorycapacity is normal.17. A method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining in a biologicalsample obtained from the subject at least one blood or urine parameterselected from the group consisting of (1) serum phosphate level, (2)serum vitamin D level, (3) serum ionized calcium level, (4) serum PTHlevel and (5) fractionary urinary phosphate excretion, wherein thesubject has a reduced risk for FGF23-mediated side effects if the atleast one blood or urine parameter is as defined in embodiment 9.18. A method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining in a biologicalsample obtained from the subject at least one blood parameter selectedfrom the group consisting (1) serum Bone Specific Alkaline Phosphataselevel; (2) serum Alkaline Phosphatase level, (3) serum N-terminalPropeptide of Type I Collagen (PINP) level and (4) serumCarboxy-terminal Collagen Crosslinks (CTx) level, wherein the subjecthas a reduced risk for FGF23-mediated side effects if the at least oneblood parameter is as defined in embodiment 10.19. A method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining whether the subjectis characterized by one or more and in particular all of the followingexclusion criteria:

(1) having undergone bariatric surgery;

(2) obesity;

(3) cardiac conditions with increased risks of arrhythmias;

(4) primary or secondary hyperparathyroidism;

(5) pulmonary disorders such as asthma or chronic obstructive pulmonarydisease (COPD)

(6) genetic diseases leading to hypophosphatemia such as X-linkedhypophosphatemia, autosomal dominant hypophosphatemic rickets, autosomalrecessive hypophosphatemic rickets;

(7) secondary hypophosphatemia or tumor induced hypophosphatemia;

(8) disorders of the bone, such as for example osteoporosis orosteomalacia; and

(9) being scheduled for surgery within 1 day to two months of the ironadministration, wherein the subject has a reduced risk forFGF23-mediated side effects if the subject is characterized by theabsence of one or more and in particular all of said exclusion criteria.

20. A method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining the respiratorycapacity of the subject, wherein the subject has a reduced risk forFGF23-mediated side effects if the respiratory capacity is normal.21. A combination of ferric carboxymaltose with one or more additionaldrugs for use in the treatment of iron deficiency, wherein theadditional drug is selected from the group consisting of:

(1) vitamin Ds;

(2) phosphates; and

(3) anti-FGF23 antagonistic antibodies.

22. The combination of embodiment 21, wherein the vitamin D iscalcitriol, alfacalcidol, cholecalciferol or ergocalciferol.23. The combination of embodiment 22, wherein calcitriol or alfacalcidolis administered within three days prior to the administration of thefirst FCM dose.24. The combination of embodiment 22, wherein administration ofcalcitriol or alfacalcidol is started on the same day of first FCM dose.25. The combination of embodiment 22, wherein calcitriol or alfacalcidoladministration is started on day 1, day 2, day 3, day 4, day 5, day 6 orday 7, or day 14 after the administration of the first FCM dose.26. The combination of embodiment 22, wherein cholecalciferol orergocalciferol is administered for a period of 14 days, 7 days, 6 days,alternatively 5 days, 4 days, 3 days, 2 days, 1 day prior toadministration of the first FCM dose.27. The combination of embodiments 22, wherein cholecalciferol orergocalciferol is administered prior to administration of the first FCMdose followed by treatment with calcitriol or alfacalcidol subsequent toadministration of the first FCM dose.28. The combination of any one of embodiments 21-27, wherein theadditional drug is administered orally.29. The combination of any one of embodiment 21-28, wherein theadditional drug is administered prior to the second dose of ferriccarboxymaltose administration.30. The combination of embodiments 21-29, for use in a method of any oneof embodiments 1-13.

The present disclosure is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, Genbank sequences, patents and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

EXAMPLES

Two prospective randomized, open-label, comparative trials wereperformed comparing the incidence of hypophosphatemia in relation totreatment with iron isomaltoside (“IIM”, tradename Monofer®,Monoferric®) and ferric carboxymaltose (“FCM”, trade name Injectafer,Ferinject) in adult human subjects with iron deficiency anaemia.

Trial Design

The trial was a randomized, open-label, comparative trial. Subjects withIron Deficiency Anaemia (IDA) were randomized 1:1 to one treatmentcourse of one of the following treatments:

-   -   Group A: iron isomaltoside 1000 (Monofer, Pharmacosmos, Holbæk,        Denmark, termed iron isomaltoside in the following), 1000 mg at        baseline    -   Group B: ferric carboxymaltose (Ferinject/Injectafer, Vifor Inc,        Switzerland), 750 mg at baseline and day 7, cumulative dose:        1500 mg

FCM was administered in two single doses of 750 mg of elemental iron oneweek apart according to its US label. Iron isomaltoside 1000 wasadministered as a single dose of 1000 mg of elemental iron.

Objectives

The primary objective of the trial was to compare the incidence ofhypophosphatemia in subjects with IDA treated with iron isomaltoside orferric carboxymaltose.

The secondary safety objective of the trial was to compare the effectsof iron isomaltoside and ferric carboxymaltose treatment in subjectswith IDA on the following:

-   (1) Incidence of severe hypophosphatemia-   (2) Time with hypophosphatemia-   (3) Proportion of subjects with hypophosphatemia at the last visit-   (4)S-phosphate (absolute [A] and relative [%] changes)-   (5) Fractional phosphate urinary excretion-   (6) Intact Fibroblast Growth Factor 23 (iFGF23), C-terminal FGF23    (cFGF23), vitamin D (25, 1,25, 24,25), Parathyroid Hormone PTH), and    ionized calcium-   (7) Adverse Events (AEs) and biochemical safety parameters

The secondary efficacy objective of the trial was to compare the effectsof iron isomaltoside and ferric carboxymaltose treatment in subjectswith IDA on Haemoglobin (Hb), s-ferritin, and Transferrin Saturation(TSAT).

In addition to the primary and secondary objectives, exploratoryanalyses on the effect of iron isomaltoside and ferric carboxymaltosewere performed including the following

-   (1) Biochemical bone/muscle markers-   (2) Muscle strength

Endpoints

The primary endpoint was the incidence of hypophosphatemia (defined ass-phosphate <2 mg/dL) at any time from baseline to day 35.

The secondary safety endpoints were the following:

-   -   Incidence of s-phosphate below 1.0 mg/dL at any time from        baseline to day 35    -   Time with hypophosphatemia (i.e. time with s-phosphate <2.0        mg/dL) from baseline to day 35    -   Proportion of subjects with hypophosphatemia at day 35    -   Absolute [Δ] and relative [%] changes in s-phosphate from        baseline to 1, 7, 8, 14, 21, and 35    -   Fractional phosphate urinary excretion at 1, 7, 8, 14, 21, and        35    -   Change in iFGF23, cFGF23, vitamin D (25, 1,25, 24,25), PTH, and        ionized calcium from baseline to 1, 7, 8, 14, 21, and 35    -   Type and incidence of AEs    -   Serious or severe hypersensitivity reaction starting on or after        the first dose of randomized treatment (i.e. treatment        emergent). The hypersensitivity terms were defined as        standardised Medical Dictionary for Regulatory Activities query        (SMQ) terms.

In addition, physical examinations and measurements of vital signs,height, weight, electrocardiogram (ECG), and safety laboratoryparameters were measured as part of standard safety assessments.

The secondary efficacy endpoints were the following:

-   -   Change in Hb, s-ferritin, and TSAT from baseline to day 1, 7, 8,        14, 21, and 35

The exploratory endpoints were the following:

-   -   Change in biochemical bone/muscle markers (serum N-terminal        Propeptide of Type I Collagen (PINP), Carboxy-terminal Collagen        Crosslinks (CTx), s-alkaline phosphatase (bone specific and        total), and creatine kinase) from baseline to day 1, 7, 8, 14,        21, and 35    -   Change in fatigue symptoms from baseline to day 14 and 35        measured by the Functional Assessment of Chronic Illness Therapy        (FACIT) Fatigue Scale    -   Change in QoL from baseline to day 14 and 35 measured by Short        Form (SF)-36 questionnaire    -   Change in bone pain from baseline to day 14 and 35 measured on a        Visual Analogue Scale (VAS)    -   Change in muscle strength from baseline to day 14 and 35        measured by grip strength    -   Change in upper and lower limb proximal muscle function from        baseline to day 14 and 35 measured by the “1 kg arm lift” test        and the “30 sec chair stand” test.    -   Change in respiratory muscles strength from baseline to day 14        and 35 measured by Maximal Inspiratory Pressure (MIP) and        Maximal Expiratory Pressure (MEP)

Safety Assessments

The trial included the following safety assessments:

-   -   Measurements of s-phosphate (blood and urine), iFGF23, cFGF23,        vitamin D (25, 1,25, 24,25), PTH, and ionized calcium    -   AEs will be collected and evaluated for relatedness, severity,        seriousness, and expectedness. They will be reported to        authorities and followed-up according to international and local        requirements    -   Physical examinations, measurements of vital signs, ECG, height,        weight, and safety laboratory parameters

Efficacy Assessments

The trial included the following efficacy assessments:

-   -   Hb, s-ferritin, TSAT, and s-iron Exploratory assessments

The exploratory assessments included the following:

-   -   Measurement of serum N-terminal PINP, CTx, s-alkaline        phosphatase (bone specific and total), and creatine kinase    -   MIP and MEP

Trial Duration and Number of Visits

For the individual subject, duration of the trial was 5 weeks (includinga 28 days screening period) and each subject attended 8 visits.

Subject Population

Subjects, who fulfilled the following eligibility criteria, wereincluded.

Inclusion Criteria:

A subject will be eligible for inclusion in the trial if he/she fulfilsthe following criteria:

-   -   1. Men or women >18 years having IDA caused by different        aetiologies* such as abnormal uterine bleeding, gastrointestinal        diseases, cancer, bariatric procedures (gastric bypass        operations), and other conditions leading to significant blood        loss    -   2. Hb≤11 g/dL    -   3. Body weight >50 kg    -   4. S-ferritin 100 ng/mL    -   5. Estimated Glomerular Filtration Rate (eGFR)≥65 mL/min/1.73 m²    -   6. S-phosphate >2.5 mg/dL    -   7. Documented history of intolerance or unresponsiveness to oral        iron therapy** for at least one month*** prior to trial        enrolment    -   8. Willingness to participate and signing the Informed Consent        Form (ICF)    -   *The aetiology (also if unknown) for IDA was documented in the        medical history and verified in the source documents.    -   **The intolerance and non-response to oral iron treatment was        documented with sign and symptoms in the medical history and        verified in the source document.    -   ***There was a documentation of intolerance or unresponsiveness        to at least one month of prescribed oral iron therapy per        investigator's judgment within the last 9 months and they would        not be candidates for oral iron again.

Exclusion Criteria:

A subject was not eligible for inclusion in this trial if he/shefulfilled any of the following criteria:

-   -   1. Acute bleeding >500 mL within 72 hours    -   2. Anaemia predominantly caused by factors other than IDA        according to Investigator's judgment    -   3. Hemochromatosis or other iron storage disorders    -   4. Known hypersensitivity reaction to any component of iron        isomaltoside or ferric carboxymaltose    -   5. Previous serious hypersensitivity reactions to any IV iron        compounds    -   6. Treatment with IV iron within the last 30 days prior to        screening    -   7. Treatment with erythropoietin or erythropoietin-stimulation        agents, red blood cell transfusion, radiotherapy, and/or        chemotherapy within the last 30 days prior to screening    -   8. Received an investigational drug within the last 30 days        prior to screening    -   9. Planned surgical procedure within the trial period    -   10. Alanine Aminotransferase (ALAT) and/or Aspartate        Aminotransferase (ASAT)>3 times upper limit of normal (e.g.        decompensated liver cirrhosis or active hepatitis)    -   11. Surgery under general anaesthesia within the last 30 days        prior to screening    -   12. Any non-viral infection within the last 30 days prior to        screening    -   13. Alcohol or drug abuse within the past 6 months    -   14. Untreated hyperparathyroidism    -   15. Kidney transplantation    -   16. Estimated life expectancy of <6 months or, for cancer        patients, an Eastern Cooperative Oncology Group (ECOG)        performance status >1    -   17. Conditions that interfere with the subject's ability to        understand the requirements of the trial and/or presumable        non-compliance    -   18. Any other laboratory abnormality, medical condition, or        psychiatric disorders which, in the opinion of the Investigator,        will put the subject's disease management at risk or may result        in the subject being unable to comply with the trial        requirements    -   19. Pregnant or nursing women. In order to avoid pregnancy,        women of childbearing potential have to use adequate        contraception (e.g. intrauterine devices, hormonal        contraceptives, or double barrier method) during the whole trial        period and 7 days after the last dosing

A summary of the subject population included is provided in thefollowing table:

IDA04 IDA05 Iron Ferric Iron Ferric Isomaltoside CarboxymaltoseIsomaltoside Carboxymaltose Safety Analysis Set (N, %) 63 (100.0) 60(100.0) 62 (100.0) 57 (100.0) Age (years) Mean (SD) 43.9 (10.4) 46.3(11.6) 42.2 (12.9) 43.1 (11.5) Median 44.0 45.5 41.0 44.0 Min-Max 25-7427-77 19-79 20-76 Sex (N, %) Female 61 (96.8) 57 (95.0) 58 (93.5) 54(94.7) Male 2 (3.2) 3 (5.0) 4 (6.5) 3 (5.3) Race (N, %) White 38 (60.3)38 (63.3) 28 (45.2) 29 (50.9) Asian 2 (3.2) 1 (1.7) Black or AfricanAmerican 22 (34.9) 19 (31.7) 32 (51.6) 27 (47.4) Other 1 (1.6) 2 (3.3) 1(3.2) 1 (1.8)

Trial Treatment

The subjects were dosed with either one treatment course of ironisomaltoside (group A) or one treatment course of ferric carboxymaltose(group B) as described below.

-   -   Group A: iron isomaltoside was administered as a single IV        infusion of 1000 mg at baseline diluted in 100 mL 0.9% sodium        chloride and given over approximately 20 minutes (50 mg        iron/min, cumulative dose: 1000 mg).    -   Group B: ferric carboxymaltose was administered as 750 mg        infused over at least 15 minutes at baseline and day 7        (cumulative dose: 1500 mg).

No premedication (e.g. antihistamine or steroids) was allowed beforeadministration of the trial drug. If the subject was in daily treatmentfor e.g. allergy or asthma this was not considered as “premedication”and could be continued.

Statistical Analyses

The primary endpoint, incidence of hypophosphatemia (defined ass-phosphate <2 mg/dL) at any time from baseline to day 35, was tabulatedand exact 95% CI were estimated for each treatment group.

Iron isomaltoside was compared to ferric carboxymaltose by estimation ofthe risk difference and the associated 95% CI, adjusting for strata(type of underlying disease (women with IDA due to gynaecological bloodlosses; yes/no) and screening s-phosphate level (< or ≥3.5 mg/dL)) usingthe Cochran-Mantel-Haenszel method.

As to sensitivity, the treatment groups were compared between thetreatment groups by a logistic regression model with treatment and typeof underlying disease as factors and baseline s-phosphate as covariateand by Fisher's exact tests.

All subjects in the safety analysis set were included in the analysis.The first post-baseline phosphate measurement was taken at day 1; hence,very few missing values are expected. If there were subject(s), for whomno post-baseline phosphate measurement(s) were available, these subjectswill be set as having s-phosphate <2 mg/dL in the primary analysis.

All the statistical analyses will be described in a statistical analysisplan.

Baseline Assessment

IDA04 IDA05 Iron Ferric Iron Ferric Isomaltoside CarboxymaltoseIsomaltoside Carboxymaltose N (%) N (%) N (%) N (%) Disease stratum: IDAdue to gynaecological blood losses Yes 41 (65.1) 42 (70.0) 44 (71.0) 39(68.4) No 22 (34.9) 18 (30.0) 18 (29.0) 18 (31.6) Disease stratum:Screening s-phosphate level  <3.5 mg/dL 32 (50.8) 33 (55.0) 35 (56.5) 32(56.1) ≥3.5 mg/dL 31 (49.2) 27 (45.0) 27 (43.5) 25 (43.9)

Trial Assessments Demographic and Baseline Assessments

Date of birth, gender, race, ethnicity, and smoking habits werecollected. A current smoker was defined as a subject who had beensmoking within the last 6 months.

Pregnancy Test

A urine pregnancy test was performed for all women of childbearingpotential. The test was handled and interpreted by the site personnel.

Relevant Medical History

Relevant medical history was recorded. Changes in medical hi story wererecorded at the subsequent visits during the trial (worsening ofsymptoms or diseases were recorded as AEs). The following was collected:disease and start and stop date. Except for underlying disorder causingIDA, start dates occurring >12 months before the enrolment into thetrial were set as >12 months.

Concomitant Medication

If the subject was receiving any concomitant medication it was recordedat the baseline visit. Changes in concomitant medication were recordedin the subsequent visits during the trial. The following was collected:brand name, indication, route, dose, frequency, unit, and start and stopdate. Start dates occurring >12 months before the enrolment into thetrial were set as >12 months.

Physical Examination

A physical examination was performed based upon the Investigator'sjudgement and could include the following:

-   -   Head-Eyes-Ear-Nose-Throat    -   Cardiovascular system    -   Respiratory system    -   Nervous system    -   Gastrointestinal system    -   Musculo-skeletal system    -   Urogenital system    -   Dermatology system    -   Others, if required

Height

Height was measured without shoes.

Weight

Weight was measured.

Vital Signs

Heart rate and blood pressure were measured at the following time pointswhen a subject received trial drug: approximately 0-10 minutes beforeinfusion, during infusion, 5-15 minutes, and 20-40 minutes after theinfusion has ended. If vital signs were measured more than once in thegiven time interval, the lowest measurement of diastolic blood pressure(including the attendant systolic blood pressure and heart rate) for theperiod was noted in the electronic Case Report Form (eCRF).

Electrocardiogram

A standard 12 lead ECG was recorded (including date, time, andsignature). At baseline and other treatment visits, two ECGs wererecorded; one before administration of the trial drug and oneapproximately 30 minutes after start of the dosing. Only one ECG wasrecorded at the follow-up visits.

The ECGs did not need to be evaluated by a cardiologist.

Respiratory Muscles Strength

The measurements of MIP and MEP provided a non-invasive clinical methodfor evaluating the strength of respiratory muscles, and it was the mostwidely used test to assess muscle pressures [ATS/ERS statement, 2002].The MIP reflected the strength of the diaphragm and other inspiratorymuscles, while the MEP reflects the strength of the abdominal musclesand other expiratory muscles. MIP and MEP were measured by MicroRPM(CareFusion Germany 234 GmbH, Hoechberg, Germany). Three tests wereperformed for both MIP and MEP with the highest value from the threetests taken as the achieved result.

Laboratory Assessments

It was requested that the blood samples were drawn before administeringthe trial drug, and, if possible, that they were drawn at the same timeof the day at all visits in order to reduce any diurnal fluctuation inthe parameters.Laboratory assessments were performed at a central laboratory. ALaboratory Manual was provided to each site in which all laboratoryprocedures were described.

Eligibility Laboratory Assessments

The following eligibility laboratory assessments were performed:

-   -   Complete haematology set: Hb, leucocytes/White Blood Cells        (WBC), erythrocytes/Red Blood Cells (RBC), haematocrit,        platelets, neutrophil granulocytes, lymphocytes, monocytes,        eosinophils, basophils, Mean Corpuscular Haemoglobin (MCH), Mean        Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin        Concentration (MCHC), and reticulocyte count    -   Biochemistry:        -   S-ferritin        -   S-phosphate        -   Alanine Aminotransferase (ALAT) and Aspartate            Aminotransferase (ASAT)        -   C-reactive Protein (CRP)        -   Estimated Glomerular Filtration Rate (eGFR)        -   PTH

Vitamin E

Vitamin E was measured at baseline visit as part of the demographicdata.

Safety Laboratory Assessments

The following safety laboratory assessments were analysed:

-   -   S-phosphate: inorganic phosphorous forms an ammonium        phosphomolybdate complex having the formula (NH4)3[PO4](MoO3)12        with ammonium molybdate in the presence of sulfuric acid. The        complex was determined photometrically in the ultraviolet region        (340 nm) of the spectrum using the Roche Modular and Cobas        Analyzer.    -   iFGF23 and cFGF23: the human intact FGF23 was measured by the        2nd generation

Elisa kit manufactured by Immonotropics, Inc, San Clemente, Calif. Thiswas a two-site enzyme-linked immunosorbent assay. The human C-terminalFGF23 was measured by the Elisa kit manufactured by Immonotropics, Inc,San Clemente, Calif. This was a two-site enzyme-linked immunosorbentassay.

-   -   Vitamin D (25, 1,25, 24,25): Following protein precipitation,        25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and their internal        standards were extracted by supported liquid extraction (SLE).        After evaporation under nitrogen, the residue was reconstituted        and analyzed using Liquid Chromatography (LC) with Tandem Mass        Spectrometric detection (MS/MS). The standard curve range was        0.5 ng/mL to 200.00 ng/mL using a serum volume of 0.1 mL.    -   PTH: The Intact PTH assay was performed using the iPTH reagent        packs for the ADVIA Centaur XP instruments. The assay was a        two-site sandwich immunoassay using direct chemiluminometric        technology, which used constant amounts of two anti-human PTH        antibodies in the Lite Reagent. The first antibody was a        polyclonal goat anti-human PTH (N-terminal 1-34) antibody        labeled with acridinium ester. The second antibody was a        biotinylated polyclonal goat anti-human PTH (39-84 region)        antibody. Streptavidin in the Solid Phase was covalently coupled        to paramagnetic latex particles. A direct relationship exists        between the amount of PTH present in the patient sample and the        amount of relative light units (RLUs) detected by the system.    -   Ionized calcium: Measured by the IL GEM Premier 3500 PAK        cartridge. The central component was the sensor card, which        provided a low volume, gas tight chamber in which the sample was        presented to the sensors. The pH and electrolyte sensors were        all based on the principle of ion-selective electrodes; that is,        an electrical potential could be established across a membrane        which was selectively permeable to a specific ion. The pH and        electrolyte sensors were polyvinyl chloride (PVC) based ion        selective electrodes, consisting of an internal Ag/AgCl        reference electrode and internal salt layer. The potentials were        measured against the card reference electrode.    -   Complete haematology set: Leucocytes/WBC, erythrocytes/RBC,        haematocrit, platelets, neutrophil granulocytes, lymphocytes,        monocytes, eosinophils, basophils, MCH, MCV, MCHC, and        reticulocyte count    -   Biochemistry:        -   S-sodium, s-potassium, s-calcium, s-urea, s-creatinine,            s-albumin        -   S-bilirubin, ASAT, ALAT        -   CRP

Efficacy Laboratory Assessments

The following efficacy laboratory parameters were analysed:

-   -   Hb    -   S-ferritin: The Access ferritin assay was a two-site        immunoenzymatic (“sandwich” assay). A sample was added to a        reaction vessel with goat anti-ferritin-alkaline phosphatase        conjugate, and paramagnetic particles coated with goat        anti-mouse: mouse anti-ferritin complexes. Serum or plasma        (heparin) ferritin binds to the immobilized monoclonal        anti-ferritin on the solid phase, while the goat anti-ferritin        enzyme conjugate reacts with different antigenic sites on the        ferritin molecules. Separation in a magnetic field and washing        removed materials not bound to the solid phase. A        chemiluminescent substrate, Lumi-Phos* 530, was added to the        reaction vessel and light generated by the reaction was measured        with a luminometer.    -   TSAT (s-iron and transferrin will be collected to calculate the        TSAT)

Exploratory Laboratory Assessments

The following exploratory laboratory assessments were analysed:

-   -   Serum N-terminal PINP: The measurement method was a sandwich        principle, electrochemiluminescence immunoassay (ECLIA). During        the first incubation, PINP in the sample and a biotinylated        monoclonal PINP-specific antibody were incubated together.        During the second incubation, streptavidin-coated labelled        microparticles and a monoclonal PINP-specific antibody labelled        with a ruthenium complex (Trs (2,2-bipyridyl)ruthenium        (II)-complex (Ru(bpy)23+)) were added to form a sandwich        complex, which bound to the solid phase via interaction of        biotin and streptavidin. The reaction mixture was aspirated into        the measuring cell where the microparticles were magnetically        captured onto the surface of the electrode. A voltage was        applied to the electrode, which induced chemiluminescent        emission, which was measured by a photomultiplier. Results were        determined via a calibration curve, which was        instrument-specifically generated by a 2-point calibration and a        master curve provided via the reagent barcode. The method was        run on a Cobas e601 Analyzer.    -   CTx: During the first incubation, 50 μL of sample and        biotinylated monoclonal anti-beta-CrossLaps antibody were        incubated together. During the second incubation,        streptavidin-coated labelled microparticles and a monoclonal P        beta-CrossLaps-specific antibody labelled with ruthenium complex        were added to form a sandwich complex, which bound to the solid        phase via interaction of biotin and streptavidin. The reaction        mixture was aspirated into the measuring cell where the        microparticles were magnetically captured onto the surface of        the electrode. Unbound substances were then removed with        ProCell. A voltage was applied to the electrode, which induced        chemiluminescent emission, which was measured by a        photomultiplier. Results were determined via a calibration        curve, which was instrument-specifically generated by a 2-point        calibration and a master curve provided via the reagent barcode.        The method was run on a Cobas e601 Analyzer.    -   S-alkaline phosphatase (bone specific and total): Bone Specific        Phosphatase (BAP) was measured on Beckman Dxi 800. The        Beckman-Coulter Ostase assay was a one-step immunoenzymatic        chemiluminescence assay. A mouse monoclonal antibody specific to        Bone Specific Alkaline phosphatase (BAP) was added to a reaction        vessels with paramagnetic particles coated with goat anti-mouse        polyclonal antibody. Calibrators, controls, and samples        containing BAP were added to the coated particles and bound to        the anti-BAP monoclonal antibody. Chemiluminescent substrate,        Lumi-Phos*530, was added to the reaction vessel and light        generated by the reaction was measured with a luminometer. The        light production is directly proportional to the amount of BAP        in the sample. The amount of BAP in the sample was determined by        means of a stored multi-point calibration curve. The level of        total Alkaline Phosphatase in blood serum was measured using a        Roche Modular Analyzer. In the presence of magnesium and zinc        ions, p-nitrophenyl phosphate was cleaved by phosphatase into        phosphate and p-nitrophenol. The p-nitrophenol released is        proportional to the ALP activity and was measured        photometrically.    -   Creatine kinase: The creatine kinase (CK) assay was performed on        the Roche Modular and Cobas Analyzers. The reaction proceeded as        follows:        -   Creatine phosphate+ADP CK >creatine+ATP        -   ATP+glucose HK >ADP+G-6-P        -   G-6-P+NADP+>G-6-PDH 6-PG+NADPH+H+    -   The formation of NADPH proceeded at the same rate as the        formation of creatine in equimolar amounts. The rate of NADPH        formation is proportional to CK activity and is measured        photometrically.

Urine Assessments

A spot urine sampling was collected in order to assess the level offractional 5-phosphate excretion.

Adverse Events

AEs were collected and evaluated for relatedness to trial drug,seriousness, severity, and expectedness.

Results

Key findings include a clear impact of the FCM dosing regimen on theendocrine system managing phosphate, vitamin-D and calcium.

The rate of hypophosphatemia was significantly higher for FCM vs TIM,both in terms of the moderate to severe form with serum phosphate below2 mg/dL and the severe form of serum phosphate at or below 1.0 mg/dL.See FIGS. 1 to 5.

Intact iFGF23 (iFGF23) increased sharply after administration of FCM andthen gradually declined over a matter of days. On the secondadministration, iFGF23 again increased, but this time to a level severalfold higher than the initial level, i.e. a surprising and previouslyunknown self-amplifying effect. For IIM essentially no change of theiFGF23 level was observed. See FIGS. 6 and 7. C-terminal FGF23 (cFGF23)which was initially high due to the underlying iron deficiency anemiadropped sharply on the first administration of FCM whereas it increasedon the second administration of FCM. See FIG. 8. Fractional urinaryexcretion of phosphate was significantly increased for FCM vs IIM. SeeFIG. 10. FCM led to an increase in PTH, a decrease in1,25-dihydroxyvitamin D, an increase in 24,25-dihydroxyvitamin D and adecrease in ionized calcium. See FIGS. 11 to 13.

Moreover, FCM led to the significant changes in bone turnover and musclefunction as measured by the following biochemical bone/muscle markers.

FCM induced a statistically significant increase of Bone SpecificAlkaline Phosphatase compared to IMM. See FIG. 14.

FCM induced statistically significant lower N-terminal PINP values thanIMM. See FIG. 14.

FCM induced a statistically significant lower CTx values than IMM. SeeFIG. 14.

FCM induced a statistically significant increase of Alkaline Phosphatasecompared to IMM. See FIG. 14.

FCM also lead to reduced muscle function relative to the comparator IViron treatment as specifically measured through respiratory capacitymeasured as maximal respiratory pressure and/or maximal inspiratorypressure. See FIG. 15.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments disclosed herein. Such equivalents are intended to beencompassed by the following claims.

NON-PATENT PUBLICATIONS

-   Aksan et al., Aliment Pharmacol Ther 2017, 45(10), 1303-1318-   Bager et al., Br J Clin Pharmacol 2017, 83, 1118-1125-   Bregman et al., Ther Adv Hematol 2014, 5(2), 48-60-   Charytan et al., Nephrol Dial Transplant 2013, 28, 953-964-   Evstatiev, Gastroenterology 2011, 141, 846-853-   Hussain et al., Anemia 2013, Article ID 169107, 10 pages-   Ikuta etal., Int J Hematol 2018,    https://doi.org/10.1007/s12185-018-2501-8-   Klein et al. BMJ Case Rep 2018; doi:10.1136/bcr-2017-222851-   Prats et al., BMC Nephrology 2013, 14:167-   Qunibi et al., Nephrol Dial Transplant 2011, 26, 1599-1607-   Sari et al., Neth J Med 2017, 75(2), 65-73-   Schaefer et al., Gastroenterology 2017, 152(6), e5-e6-   Seid et al., Am J Obstet Gynecol 2008, 199:435.e1-435.e7-   Stein et al., Scand J Gastroenterol 2018,    https://doi.org/10.1080/00365521.2018.1498914-   Van Wyck et al., Transfusion 2009, 49(12), 2719-2728-   Wolf et al., Journal of Bone and Mineral Research 2013, 28(8),    1793-1803-   Zoller et al., Curr Opin Nephrol Hypertens 2017, 26(4), 266-275

1-15. (canceled)
 16. A method of treating iron deficiency, whichcomprises administering ferric carboxymaltose to a subject, wherein thesubject has a reduced risk for FGF23-mediated side effects.
 17. Themethod of claim 16, wherein the subject having a reduced risk forFGF23-mediated side effects has a blood or urine parameter selected fromthe group consisting of: (1) normal serum phosphate level, inparticular >2.5 mg/dL; (2) normal serum vitamin D level, in particular1,25-dihydroxyvitamin D level being within the following ranges: Males:≤16 years: 24-86 pg/mL, ≥16 years: 18-64 pg/mL, Females: <16 years:24-86 pg/mL, ≥16 years: 18-78 pg/mL; (3) normal serum ionized calciumlevel, in particular 1.16-1.32 mg/dL; (4) normal serum PTI-1 level, inparticular 15-65 pg/mL; (5) normal fractionary urinary phosphateexcretion, in particular an FEPi of 10-20% (0.1-0.2 fraction); and (6) acombination of (1), (2), (3), (4) and (5).
 18. The method of claim 16,wherein the subject having a reduced risk for FGF23-mediated sideeffects has a blood parameter selected from the group consisting of: (1)normal serum Bone Specific Alkaline Phosphatase level, in particular6.5-22.4 U/L; (2) normal serum Alkaline Phosphatase level, in particular31-40 U/L; (3) normal serum N-terminal Propeptide of Type I Collagen(PINP) level, in particular 15.13-85.50 ng/mL; (4) normal serumCarboxy-terminal Collagen Crosslinks (CTx) level, in particular0.03-1.01 ng/mL; and (5) a combination of (1), (2), (3) and (4).
 19. Themethod of claim 16, wherein the subject having a reduced risk forFGF23-mediated side effects is characterized by the absence of one ormore and in particular all of the following exclusion criteria: (1)having undergone bariatric surgery; (2) obesity; (3) cardiac conditionswith increased risks of arrhythmias; (4) primary or secondaryhyperparathyroidism; (5) pulmonary disorders; (6) asthma or chronicobstructive pulmonary disease (COPD); (7) genetic diseases leading tohypophosphatemia; (8) X-linked hypophosphatemia, autosomal dominanthypophosphatemic rickets, autosomal recessive hypophosphatemic rickets;and (9) secondary hypophosphatemia or tumor induced hypophosphatemia;(10) disorders of the bone; (11) osteoporosis or osteomalacia; and (12)being scheduled for surgery within 1 day to two months of the ironadministration.
 20. The method of claim 16, wherein the subject having areduced risk for FGF23-mediated side effects is characterized by normalrespiratory capacity measured as maximal respiratory pressure and/ormaximal inspiratory pressure, in particular a maximal respiratorypressure of: Males: ≥117−(0.83×age) cm H₂O, Females: ≥95−(0.57×age) cmH₂O; and/or a maximal inspiratory pressure of: Males: ≥62−(0.15×age) cmH₂O, Females: ≥62−(0.50×age) cm H₂O.
 21. A method of monitoring asubject who has been administered a dose of ferric carboxymaltose,comprising determining in a biological sample obtained from the subjectat least one blood or urine parameter selected from the group consistingof (1) serum phosphate level, (2) serum vitamin D level, (3) serumionized calcium level, (4) serum PTH level and (5) fractionary urinaryphosphate excretion, wherein the subject is eligible for beingadministered a further dose of ferric carboxymaltose if the at least oneblood or urine parameter is as defined in claim
 17. 22. A method ofmonitoring a subject who has been administered a dose of ferriccarboxymaltose, comprising determining in a biological sample obtainedfrom the subject at least one blood parameter selected from the groupconsisting of (1) serum Bone Specific Alkaline Phosphatase level; (2)serum Alkaline Phosphatase level, (3) serum N-terminal Propeptide ofType I Collagen (PINP) level and (4) serum Carboxy-terminal CollagenCrosslinks (CTx) level, wherein the subject is eligible for beingadministered a further dose of ferric carboxymaltose if the at least oneblood parameter is as defined in claim
 18. 23. A method of monitoring asubject who has been administered a dose of ferric carboxymaltose,comprising determining the respiratory capacity of the subject, whereinthe subject is eligible for being administered a further dose of ferriccarboxymaltose if the respiratory capacity is normal.
 24. The method ofclaim 21 wherein the dose is 750 mg of elemental iron and the dose is750 mg of elemental iron.
 25. The method of claim 22 wherein the dose is750 mg of elemental iron and the dose is 750 mg of elemental iron. 26.The method of claim 23, wherein the dose is 750 mg of elemental iron andthe dose is 750 mg of elemental iron.
 27. A method of identifying asubject having a reduced risk for FGF23-mediated side effects,comprising determining in a biological sample obtained from the subjectat least one blood or urine parameter selected from the group consistingof (1) serum phosphate level, (2) serum vitamin D level, (3) serumionized calcium level, (4) serum PTH level and (5) fractionary urinaryphosphate excretion, wherein the subject has a reduced risk forFGF23-mediated side effects if the at least one blood or urine parameteris as defined in claim
 17. 28. A method of identifying a subject havinga reduced risk for FGF23-mediated side effects, comprising determiningin a biological sample obtained from the subject at least one bloodparameter selected from the group consisting (1) serum Bone SpecificAlkaline Phosphatase level; (2) serum Alkaline Phosphatase level, (3)serum N-terminal Propeptide of Type I Collagen (PINP) and (4) serumCarboxy-terminal Collagen Crosslinks (CTx) level, wherein the subjecthas a reduced risk for FGF23-mediated side effects if the at least oneblood parameter is as defined in claim
 18. 29. A method of identifying asubject having a reduced risk for FGF23-mediated side effects,comprising determining whether the subject is characterized by one ormore and in particular all of the following exclusion criteria: (1)having undergone bariatric surgery; (2) obesity; (3) cardiac conditionswith increased risks of arrhythmias; (4) primary or secondaryhyperparathyroidism; (5) pulmonary disorders; (6) asthma or chronicobstructive pulmonary disease (COPD); (7) genetic diseases leading tohypophosphatemia; (8) X-linked hypophosphatemia, autosomal dominanthypophosphatemic rickets, autosomal recessive hypophosphatemic rickets;and (9) secondary hypophosphatemia or tumor induced hypophosphatemia;(10) disorders of the bone; (11) osteoporosis or osteomalacia; and (12)being scheduled for surgery within 1 day to two months of the ironadministration, wherein the subject has a reduced risk forFGF23-mediated side effects if the subject is characterized by theabsence of one or more and in particular all of said exclusion criteria.30. A method of identifying a subject having a reduced risk forFGF23-mediated side effects, comprising determining the respiratorycapacity of the subject, wherein the subject has a reduced risk forFGF23-mediated side effects if the respiratory capacity is normal.